Peripheral-type benzodiazepine receptor expression level as an index of organ damage and regeneration

ABSTRACT

The present invention relates to methods, reagents, and kits for assessing organ damage, such as damage due to ischemia reperfusion injury, in the course of a transplantation therapy and/or for assessing organ regeneration following transplantation therapy. The invention provides a method for determining an index of organ health in the course of transplantation therapy comprising measuring the expression level of peripheral-type benzodiazepine receptor (PBR) in the organ. Measuring the expression level of PBR is also useful for assessing the progress of organ regeneration in the course of transplantation therapy by comparing the index of organ health. The expression level of PBR may be used as a predictor of the outcome of transplantation therapy.

CROSS-REFERENCE TO EARLIER APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/512,060, filed Dec. 8, 2005, now allowed, which is a U.S. NationalStage of PCT/US 2003/12385, filed Apr. 22, 2003, which claims thebenefit of U.S. Provisional Application No. 60/374,136, filed Apr. 22,2002, each earlier application is hereby expressly incorporated byreference in its entirety and is assigned to the assignee hereof.

GOVERNMENT FUNDING

Work described herein was supported in part by funding from the NationalInstitute of Health. The United States Government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods, reagents, and kits forassessing organ damage, such as damage due to ischemia reperfusioninjury, in the course of a transplantation therapy and/or for assessingorgan regeneration following transplantation therapy.

2. Description of the Related Art

The central focus of organ transplantation therapy has been theprevention of acute rejection. (See for example, The Handbook ofTransplantation Management by Leonard Makowka, CRC Press, 1991.) Inattempting to prevent acute rejection, therapies have evolved to reduceor to control the appropriate recognition of allopeptides by helper Tcells. As a result, early rates of acute rejection have lowered to below20% and increased one-year renal allograft survival to well above 80%.Unfortunately, this improved early graft survival has not translated toimproved long-term graft survival. Graft half-life and the effects ofchronic allograft nephropathy have remained relatively constantthroughout the eras of calcineurine inhibition and monoclonal antibodytherapies.

Survival of any organ or cell is dependent on the availability of oxygenand crucial nutrients and removal of cellular waste. Metabolic processesmust function in a balanced manner to maintain cellular homeostasis.Disruption of this critical balance by physical, chemical or oxidativestress results in changed rates and direction of normal biochemical andmolecular reactions as the cells attempt to maintain cell functions andadapt to stressful conditions.

Oxidative stress is induced by the total stoppage of blood flow(ischemia) incident to the removal of an organ from a donor, coldstorage, warming, and re-implantation into a recipient in the course oftransplantation and results in a fundamental metabolic imbalance.Switching from aerobic to anaerobic conditions results in anaccumulation of harmful substrates and stimulation of catabolic pathwaysto eliminate undesirable metabolic byproducts. This metabolic imbalancecontinues as long as a lack of oxygen lasts. Ultimately, diminishedmetabolic rates, metabolic acidosis and calcium and sodium overloadingaccelerate cell and organ death. Reperfusion, resumption of oxygenatedblood flow to an ischemic organ, intensifies injury by providingconditions that activate free radical production and a cascade ofreactions leading to recruitment and activation of neutrophils andplatelets. Organ damage caused in this process is known as ischemiareperfusion injury (IRI).

With respect to kidney transplant, it is known that cold storage maycause delayed graft function (DGF), which in turn causes reduced shortand long term renal allograft survival (Ojo, et al., Transplantation,63:968-974, 1997). However, while prolonged cold ischemia is known tohave detrimental effects on graft survival, the cellular and molecularresponses of the kidney to ischemic insult are not completelyunderstood. Various experimental studies have tried to determine themechanisms involved in acute ischemic renal failure. (See, for example,a review by Sheridan and Bonventre (Curr. Opin. Nephrol. Hypertension,9:427-34, 2000).

Its remarkable regeneration potential enables the kidney to completelyrestore its function and to replace damaged cells and to restoreepithelial continuity. However, in different situations, recovery isdelayed or does not occur at all. The mechanism underlying thedetrimental effect of cold ischemia and thermal injury on graft survivalremains unclear and the factors that trigger and control the repairprocess are poorly understood. For background on kidney diseaseincluding theory and practice known to one of skill in the art, seeDisease of the Kidneys (6^(th) Ed.) by Robert Schrier and CarlGottschalk, Little Brown and Company (1996).

Currently, assessment of renal graft dysfunction followingtransplantation relies on the measurement of plasma creatinine and theoften inconclusive histological results of a renal biopsy. In addition,the results of such measurements are essentially descriptive and are noteffective predictive markers.

Therefore, the art is in need of improved methods for the assessment oforgan health in the course of transplantation therapy, for outcomeprediction, and for the assessment of regeneration followingtransplantation therapy. These and other objects are provided by thepresent invention.

SUMMARY OF THE INVENTION

The invention provides a method for determining an index of organ healthin the course of transplantation therapy comprising measuring theexpression level of peripheral-type benzodiazepine receptor (PBR) insaid organ whereby said index is determined.

The invention also provides for the method further comprising obtaininga tissue sample of the organ.

The invention further provides for the method wherein the organ isselected from among one or more of kidney, heart, lung, cornea, skin,liver, bone marrow, vascular graft, pancreas, and small bowel.

The invention further provides for the method wherein the organ is akidney.

The invention further provides for the method wherein the index ofhealth is an index of ischemia reperfusion injury.

The invention further provides for the method wherein the index of organhealth is an index of ischemia reperfusion injury incident to a renaltransplant procedure.

The invention further provides for the method wherein the expressionlevel of peripheral-type benzodiazepine receptor is measured using amethod chosen from one or more of immunohistochemistry, electrophoreticblotting, hybridization of a nucleic acid probe to mRNA, observingbinding of radiolabeled ligand, observing binding of fluorescencelabeled ligand, observing binding of isotopically labeled ligand, and invivo scanning using isotopically labeled ligand of PBR.

The invention further provides for the method wherein the index of organhealth is determined at one or more points in the course oftransplantation therapy selected from among the time of organextraction, the time of organ storage, immediately prior to organimplantation, following reperfusion in the recipient, and at one or moreintervals following the transplantation procedure.

The invention further provides a method for assessing the progress oforgan regeneration in the course of transplantation therapy by comparingthe index of organ health, determined according to a method set forthabove, to normalized data of the index of organ health.

The invention further provides a method of predicting the outcome oftransplantation therapy wherein the index of organ health is determinedby the method set forth above and comprising the step of comparing saidindex with a correlation between said index and previous outcomeswhereby a likely outcome is predicted.

The invention further provides a method of assessing transplant therapyprocedures comprising determining an index of organ health according toa method set forth above and comparing index measurements as a functionof parameters of said transplant therapy procedures.

The invention further provides a kit of materials and/or reagent(s) forthe practice of the method of the invention.

The invention further provides such a kit of materials and/or reagent(s)for the practice of the method of the invention comprising an indicatorof PBR expression selected from among an antibody to PBR, a nucleic acidprobe capable of specific hybridization to mRNA encoding PBR, and aradiolabeled, isotopically labeled, or fluorescence labeled ligand ofPBR.

The invention further provides such a kit of materials and/or reagent(s)for the practice of the method of the invention further comprisingcontrol and or comparative samples.

The invention further provides a kit of materials and reagent(s) for thepractice of the method of the invention comprising a microanalyticalapparatus.

The invention further provides such a kit wherein the microanalyticalapparatus is a lab-on-a-chip.

The invention further provides a method for treating a patient in thecourse of transplantation therapy comprising administration of aneffective amount of a pharmaceutical composition comprising an agentwhich modulates expression of PBR.

The invention further provides a method for treating a patient in thecourse of transplantation therapy comprising administration of aneffective amount of a pharmaceutical composition comprising an agentwhich modulates the activity of PBR.

The invention further provides for such methods of treatment wherein theprogress of the treatment is monitored by measuring the index of organhealth according to a method set forth above.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and advantages of the invention will become apparent fromthe following detailed description of the preferred embodiments thereofin connection with the accompanying drawings, in which:

FIG. 1 shows the effect of cold ischemia on the glomerular filtrationrate (GFR) (A), sodium reabsorption (B), proteinuria (C) and alanineexcretion (D). Renal function was determined in control anduninephrectomized animals (Nef) (control, closed diamond and Nef, closedsquare). Autotransplanted kidneys were cold-flushed and preserved withUniversity of Wisconsin (UW) solution for 1 h (UW1 h, closed triangle),24 h (UW24 h, open square), 48 h (UW48 h, open circle) and 72 h (UW72 h,closed circle). (*P<0.05 UW1 h and UW24 h vs. Control and Nef, **P<0.01UW1 h and UW24 h vs. Control and Nef, ***P<0.001 UW1 h and UW24 h vs.Control and Nef, ^(#)P<0.05 UW48 h and UW72 h vs. Control and Nef,^(##)P<0.01 UW48 h and UW72 h vs. Control and Nef, ^(###)P<0.001 UW48 hand UW72 h vs. Control and Nef).

FIG. 2 shows the effect of cold ischemia on TMAO (A), and citrateexcretion (B). Renal function was determined in control anduninephrectomized animals (control, closed diamond and Nef, closedsquare). Autotransplanted kidneys were cold-flushed and preserved withUW solution for 1 h (UW1 h, closed triangle), 24 h (UW24 h, opensquare), 48 h (UW48 h, open circle) and 72 h (UW72 h, closed circle).(*P<0.05 UW1 h and UW24 h vs. Control and Nef, **P<0.01 UW1 h and UW24 hvs. Control and Nef, ***P<0.001 UW1 h and UW24 h vs. Control and Nef,^(#)P<0.05 UW48 h and UW72 h vs. Control and Nef, ^(##)P<0.01 UW48 h andUW72 h vs. Control and Nef, ^(###)P<0.001 UW48 h and UW72 h vs. Controland Nef).

FIG. 3 shows representative interstitial immunostaining of vimentin inautotransplanted kidneys at week 16. Kidneys cold-flushed and preservedwith UW solution for 1 h (A) or 24 h (B) for 48 h (D) or 72 h (C).Vimentin immunostaining was performed as described in the Examples.Original magnification: ×100.

FIG. 4 shows representative immunostaining of PBR in human (A and B) andpig (C) and (D) kidneys. PBR immunostaining was performed as describedin the Examples. Original magnification: ×100.

FIG. 5 shows representative immunostaining of PBR in autotransplantedpig kidneys after different times of cold preservation. Kidneyscold-flushed and preserved with UW solution for 1 h (A) after 40 to 60min of reperfusion, (B) at week 4, (C) at week 16. Kidneys cold-flushedand preserved with UW solution for 24 h (D) after 40 to 60 min ofreperfusion, (E) at week 4, (F) at week 16. Kidneys cold-flushed andpreserved with UW solution for 48 h (G) after 40 to 60 min ofreperfusion, (H) at week 4, (I) at week 16. Kidneys cold-flushed andpreserved with UW solution for 72 h (J) after 40 to 60 min ofreperfusion, (K) at week 4, (L) at week 16. PBR immunostaining wasperformed as described in the Examples. Original magnification: ×100.

FIG. 6 shows PBR staining changes in the course of kidney regeneration.Evolution of PBR immunostaining in kidneys preserved for 1 h (A and D),24 h (E), 48 h (B and F) or 72 h (C) and (G). PBR immunostaining wasperformed as described in the Examples. Original magnification: ×100.

FIG. 7 shows identification of CD4⁺ (A), CD8⁺ (B) and MCA1218⁺ cells (C)cells in post transplanted pig kidneys. Autotransplanted kidneys werecold-flushed and preserved with UW solution for 1 h (UW1 h, closedtriangle), 24 h (UW24 h, open square), 48 h (UW48 h, open circle) and 72h (UW72 h, closed circle). CD4⁺, CD8⁺ and MCA1218⁺ cells were identifiedas described in the Examples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides for the determination of an index oforgan health, damage and/or viability by measuring the expression ofperipheral-type benzodiazepine receptor (PBR). The method isparticularly useful for assessment or outcome prediction in the courseof transplantation therapy. The method is preferably used to determinean index of organ health after ischemia and reperfusion in the course oforgan transplantation therapy.

The invention is based on the discovery of the inventors that PBRexpression is correlated with ischemia reperfusion injury and recovery,and that the expression level of PBR tracks known injury parameters.Therefore, measurement of the expression level of PBR in the course oftransplantation therapy and/or subsequent to a transplant procedureprovides a basis for a method of assessing organ health, damage,recovery, and predicting the outcome of such therapy.

The determination of PBR expression levels according to the inventionmay also be utilized as part of a research method for the development oftransplantation strategies and techniques. In this aspect of theinvention, variations in transplantation strategies and techniques maybe assessed by tracking the index of organ health, damage and/orviability by measuring the expression of peripheral-type benzodiazepinereceptor (PBR).

Furthermore, the discovery forms a basis of a method of treatmentwherein agents which modulate PBR expression or activity is administeredto improve organ regeneration following a transplant procedure or otherischemic occurrence. In this aspect of the invention, a therapeuticallyeffective amount of an agent which modulates PBR expression or activityis administered to a patient in need of treatment. Such a patient wouldtypically be recovering from transplantation therapy, preferably renaltransplantation therapy, although the method may be applied to a patienthaving experienced another cause of ischemic damage.

Reagents and materials for the determination of PBR expression levelsmay be conveniently packaged as a kit for the practice of the methods ofthe invention. Such kits preferably comprise a reagent useful for themeasurement of PBR expression levels and optionally additionalmaterials, such as slides and fixing material, precast gels, secondaryantibodies, apparatus parts for use in the method, and the like.Preferably, such a kit of materials and/or reagent(s) for the practiceof the method of the invention comprises an indicator of PBR expressionselected from among an antibody to PBR, a nucleic acid probe capable ofspecific hybridization to mRNA encoding PBR, and a radiolabeled,isotopically labeled, or fluorescence labeled ligand of PBR. Such a kitof materials and/or reagent(s) may also comprise control and/orcomparative samples. In one embodiment of the invention, a kit ofmaterials and reagent(s) for the practice of the method of the inventioncomprises a microanalytical apparatus, for example, the microanalyticalapparatus may be in the form of a lab-on-a-chip apparatus. In analternative embodiment, the kit may be supplied as a lab-on-a-chipapparatus for use in an automated analysis system.

Mitochondria are hypothesized to play a central role in ischemiareperfusion injury because the switch of aerobic to anaerobic conditionsis one of the first steps in the ischemic process. At present, there isno specific marker to assess mitochondrial function, viability andrecovery. Previous studies have evaluated different techniques such asnuclear magnetic resonance spectroscopy. See, for example, a review byNeild et al., (Nephrol. Dial. Transplant., 12:404-17, 1997). However,this technique is generally too expensive and impractical for routineuse.

Cholesterol is a critical component of plasma membranes. Molitoris etal., have suggested that renal ischemia may cause an acute reduction inapical membrane cholesterol, relative to its total phospholipid content(Molitoris et al., J Membr. Biol., 106:233-242, 1998). Recently, Zageret al., have determined that in vivo IRI acutely increased cholesterolester (CE), but not free cholesterol (FC), content, indicating perturbedCE/FC cycling (Zager et al., Kidney Int., 59:1750-61, 2001). Otherstudies have demonstrated that when cholesterol levels are decreased incultured proximal tubular cells, tubule susceptibility to injury ismarkedly enhanced (Zager et al., Kidney Int., 56:1788-97, 1999). Thebiochemical modification of plasma membrane cholesterol either bycholesterol esterase or cholesterol oxidase treatment, in culturedproximal tubular cells, is rendered highly vulnerable to superimposedhypoxic or toxic challenges (Zager et al., supra). Exposition ofproximal tubular segments to high doses of cholesterol esterase orcholesterol oxidase induced profound mitochondrial dysfunction followedby necrotic cell death (Zager et al., Kidney Int., 58:193-205, 2000). Acorrelation between elevated cholesterol and cytoresistance by 18 to 24hours after different forms of in vivo renal injury was alsodemonstrated and correlated to proximal tubular cell resistance tosuperimposed attack (Zager et al., Kidney Int., 56:1788-97, 1999; Zageret al., Am. J. Pathol., 157:1007-16, 2000; Zager et al., Am. J. Pathol.,159:743-52, 2001).

PBR is an 18-kDa protein which was originally discovered because itbinds the benzodiazepine diazepam with relatively high affinity(Papadopoulos Endocr. Rev., 14:222-240, 1993). PBR is an indispensableelement of the cholesterol transport machinery (Papadopoulos,Proceedings of the Society for Experimental Biology & Medicine, 1998;217:130-142). This is consistent with data showing that PBR is a highaffinity cholesterol binding protein. (H. Li et al., Proceedings of theNational Academy of Sciences USA, 98:1267-1272, 2001; J. J. Lacapere etal., Biochem Biophys Res Com., 284:536-641, 2001). PBR is mainlylocalized in the outer mitochondrial membrane and was initiallydescribed as a functional component of the steroidogenic machinerymediating cholesterol delivery from the outer to the inner mitochondrialmembranes (Anholt et al., J. Biol. Chem., 261:576-83, 1986;Papadopoulos, et al., J. Biol. Chem., 265:3772-79, 1990). However, PBRis present in most tissues examined including kidney. The functionalmitochondrial PBR is a multimeric receptor complex. It is composed of atleast the 18-kDa isoquinoline binding protein organized in clusters offour to six molecules, the 34-kDa voltage-dependent anion channel, andthe adenine nucleotide carrier (Garnier et al., Mol. Pharmacol.,45:201-211, 1994). Further studies have demonstrated that targeteddisruption of the PBR gene in Leydig cells resulted in the arrest ofcholesterol transport into mitochondria and transfection of thePBR-disrupted cells with a PBR cDNA rescued cholesterol transport(Papadopoulos et al., J. Biol. Chem., 272:32129-35, 1997). From thesestudies a region of the cytosolic carboxyl terminus receptor wasidentified as a cholesterol-binding site (Li et al., Proc. Natl. Acad.Sci. USA, 98:1267-72, 2001).

Because cholesterol is a major component of plasma membrane, wehypothesized that PBR could be involved in renal reparation after coldischemia and reperfusion in an autotransplanted pig kidney model. Thereis growing evidence of the importance of PBR in the transport of thesubstrate cholesterol into mitochondria in streroidogenic (Krueger etal., J. Biol. Chem., 265:15015-22, 1990) and liver tissues (Tsankova etal., Eur. J. Pharm., 294:601-7, 1995). We used a well-establishedautotransplant pig kidney model and we have focused on the renalfunction and morphology and PBR expression.

In a model study, described in the Examples below, PBR levels weregreater in kidneys preserved at 4° C. in University of Wisconsin (UW)solution for 1 h and 24 h compared to kidneys preserved for 48 h and 72h. Interestingly, PBR immunoreactivity was associated with the lesssevere urine excretion of TMAO. One week after surgery, PBR levelsrecovered in kidneys where reparation was most efficient (1 h and 24 h).However, after week four, the intensity of the staining decreased in the24 h group and to a greater extent in the 1 h groups. The data disclosedherein is consistent with a study suggesting that PBR might be involvedin nerve degeneration and regeneration (Lacor et al., Brain Res.,815:70-80, 1999).

A significant result of the present study is the observation that coldischemia and particularly long preservation time (i.e., 24 to 72 hours)induced greater functional deterioration when compared with shorter coldischemia times such as about 1 hour. In model experiments, Na⁺ andaminoaciduria transport was dramatically impaired after coldpreservation particularly after 72 h of cold ischemia. These functionalresults were related to observed differences in tubular cells, whichwere damaged in 48 hour and 72 hour groups when compared to 1 hour and24 hour preserved groups.

Previous studies have demonstrated that natriuresis occurs as a resultof IRI, which involves a pronounced down-regulation of different Na⁺transporters and a loss of Na⁺—K⁺-ATPase pump distribution from basal toinappropriate apical location (Breton et al., J. Am. Soc. Nephrol.,9:155-66, 1998; Alejandro et al., Kidney Int., 48:1308-15, 1995; Wang etal., J. Am. Soc. Nephrol., 9:605-13, 1998; Kwon et al., Kidney Int.,55:963-75, 1999). Reductions in glomerular filtration rate (GFR) havebeen attributed to persistent vasoconstriction, activation oftubuloglomerulo feedback as a result of a high Na⁺ and solute deliveryto the macula densa, and an increase in paracellular permeabilityresulting in “back-leak” of glomerular filtrate (Trocha et al., Ann.Surg., 230:105-13, 1999). The development of significant and progressiveproteinuria is also related to chronic injury and extensive fibrosiswhere glomeruli were mostly obsolescent with focal segmental collapse ofthe capillary loops and were associated with the diffuse infiltration ofinterstitial mononuclear cells. Thus, renal medulla injury is associatedwith the time duration of cold ischemia as demonstrated by the osmoliteexcretion in urine.

The expression level of PBR may be observed by any means recognized inthe art including both in vivo and ex vivo methods. As an example, apreferred method is immunohistochemical staining and microscopicobservation of tissue samples. Using immunohistochemistry to observe PBRexpression in a pig model of IRI recovery in the course of renaltransplant, it is observed that PBR staining density increases afterIRI. Once reparation was complete, PBR staining tends to drop again, butthe staining is still detected in more than 90% of tubular sections. Bycontrast, when regeneration was incomplete or slowed, PBR expressionremains intense but limited to few tubular sections. A lack of PBRstaining as observed by immunohistochemistry is an indicator of moresevere organ damage and/or a predictor of organ failure. PBR was notdetected in interstitial fibrosis and necrotic tissue, such as in nonprimary function, i.e., where an organ does not function followingtransplantation. Therefore, the level and course of PBR expression inorgan tissues can be used to assess organ health as well as the progressof recovery from organ damage, such as organ damage caused by IRI. Theseresults indicate that measurements of PBR expression may also be used asan indicator of organ viability.

These conclusions are supported by further observations such as thecorrelation with citrate excretion. The formation of citrate from acetylcoenzyme A and oxaloacetate is catalyzed by citrate synthase, which is akey enzyme in the tricarboxylic acid cycle (Ullian et al., Hypertension,35:875-79, 2000). Citrate synthase activity is also related to themitochondrial membrane integrity. In the model of IRI in renaltransplant, in prolonged conservation groups, an observed reduction ofurinary citrate excretion is consistent with reduced renal parenchymalcitrate synthase activity and loss of mitochondrial membrane integrityduring the first weeks. Citrate excretion increased in these groupsafter week two, correlated with reparation progress. However, citrateexcretion remained at a low rate when compared to the other groups.Consequently, we observe that citrate excretion is consistent withmitochondrial viability and correlates with PBR expression, regenerationprocesses and tissue reparation.

Moreover, cold ischemia influences long-term histology and interstitialchanges in the pig kidney independently from allogenicity. Vimentinstaining is modulated by IRI. Atrophic tubules lacked a brush border,thickened basement membrane and were immunoreactive for vimentin, whichis known to be expressed by degenerating and regenerating cells(Nakatsuji et al., Virchows Arch., 433:359-67, 1998).

Furthermore, observations indicate that cellular infiltration stronglycorrelates with the intensity of renal damage. After implantation, renaldamage was significantly reduced in kidneys preserved with UW solutionfor 1 h or 24 h when compared to 48 h and 72 h preservation. The dataalso suggest that T cells play a major role in the development of renalIRI mediated probably by adhesion of infiltrating T cells to renaltubular cells. However, a role for lymphocytes in this autotransplantedpig kidney model is not immediately intuitive based on classicimmunologic paradigms. Classically, T cell activation has been thoughtto require foreign antigen bound to a self-major histocompatibilitycomplex molecule together with costimulatory signals byantigen-presenting cell. The absence of foreign antigens suggests thatalloantigen-independent T cell activation may be involved in renal IRI.Accordingly, the present observations are consistent with a recentstudy, which demonstrated that the CD4⁺ is an important mediator ofischemic injury (Burne et al., T. Clin. Invest., 108:1283-90, 2001).

PBR is known to be involved in cholesterol transport in steroidogenicorgans. However, the kidney is not a steroidogenic organ. Thus, a rolefor PBR and cholesterol transport in IRI and its long-term effect onrenal function was not obvious. However, based on the model data, itappears that PBR has a more general role in intracellular cholesteroltransport, trafficking and compartmentalization. Not wishing to be boundby theory, PBR expression is implicated as a key mediator, in a secondstep, after up-regulation of proximal tubule cholesterol content, whichcontributes to stabilization of the plasma membrane. Therefore, PBRexpression may be utilized as a more efficient and general marker ofregeneration than other markers of chronic injury.

Consistent with an important general role for PBR, and supporting thevalidity of the model system, we have observed that PBR localization inhuman kidney is very similar to pig kidney. Furthermore, the PBRsequence presents a high sequence homology (>80%) across species.Accordingly, the pig model may be used to assess new protective drug orpreservation solutions and give new insights in IRI pathophysiology,which are applicable to humans (Oke et al., Mol. Cell Endocrinol.,83:1-9, 1992). Therefore, measuring PBR expression level in tissuescomprises an improvement to animal model methods of transplant researchsuch as are described in Handbook of Animal Models in TransplantationResearch by Donald Cramer, Luis G. Podesta, Leonard Makowka, CRC Press;(1994). The molecular mechanisms that mediate this modulation of PBRremain undefined. This is a novel approach in renal transplantmanagement and can provide new insight in the mechanisms of IRI. SincePBR is localized in the same areas in the human and pig kidneys, theimproved pig model system provides a method for development of clinicalstrategies for the prevention of delayed graft function and improvementof renal reparation process.

In practicing such a method, clinical transplantation strategies andtechniques may be efficiently assessed by measuring the course of PBRexpression as a function of variation in procedural parameters ortechniques. Such a method is preferably applied in the model systemprior to further study in a clinical setting.

The expression level of PBR in sample tissue may be determined by anyappropriate method recognized in the art. Appropriate methods which willbe known and recognized in the art include, for example, immunologicalmethods and the use of radiolabeled or fluorescent ligands of PBR tomeasure the level of PBR directly, such as ELISA assays,immunohistochemistry, electrophoretic blotting and molecular biologymethods, such as expression profiling, to measure the cellular level ofmRNA encoding PBR. The observed expression levels may be compared tovalues in normal tissue and/or to time course profile values from priortherapeutic experience. The invention provides that such methods may beconducted in microscale using “lab-on-a-chip” technology. In addition,it is possible to measure PBR expression levels by isotopic labeling ofligand compounds which may be observed by in vivo scanning methods suchas magnetic resonance, e.g., MRI.

Immunohistochemical measurement of PBR expression is illustrated in theExamples. Another exemplary assay format which may be used to monitorthe expression level of PBR is measurement of mRNA expression. Forinstance, mRNA expression may be monitored directly by hybridization tonucleic acids derived from the PBR sequence. Total RNA or mRNA isisolated from tissue samples by standard procedures such those disclosedin Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,Current Protocols in Molecular Biology (Greene Publishing Co., NY,1995); Maniatis et al., Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982); and Ausubelet al., Short Protocols in Molecular Biology: A Compendium of Methodsfrom Current Protocols in Molecular Biology (1999).

Probes to detect differences in RNA expression levels between cellsexposed to the agent and control cells may be prepared from thepublished sequence of PBR. It is preferable, but not necessary, todesign probes which hybridize only with target nucleic acids underconditions of high stringency. Only highly complementary nucleic acidhybrids form under conditions of high stringency. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity which should exist between two nucleic acid strands inorder to form a hybrid. Stringency should be chosen to maximize thedifference in stability between the probe:target hybrid and potentialprobe:non-target hybrids.

Probes may be designed from the nucleic acids of the invention throughmethods known in the art. For instance, the G+C content of the probe andthe probe length can affect probe binding to its target sequence.Methods to optimize probe specificity are commonly available. (See forexample, Sambrook et al., (1989) or Ausubel et al., (Current Protocolsin Molecular Biology, Greene Publishing Co., NY, 1995).)

Hybridization conditions are modified using known methods, such as thosedescribed by Sambrook et al., (1989) and Ausubel et al., (1995), assuitable for each probe. Hybridization of total cellular RNA or RNAenriched for polyA RNA can be accomplished in any available format. Forinstance, total cellular RNA or RNA enriched for polyA RNA can beaffixed to a solid support and the solid support exposed to at least oneprobe comprising at least one, or part of one of the sequences of theinvention under conditions in which the probe will specificallyhybridize. Alternatively, nucleic acid fragments complementary to PBRmRNA can be affixed to a solid support, such as a porous glass wafer.The glass or silica wafer can then be exposed to total cellular RNA orpolyA RNA from a sample under conditions in which the affixed sequenceswill specifically hybridize. Such glass wafers and hybridization methodsare widely available, for example, those disclosed by Beattie (WO95/11755). By examining for the ability of a given probe to specificallyhybridize to an RNA sample from a normal organ and a sample from atransplant organ, relative levels of PBR expression may be determined.

Microarray technology and transcriptional profiling are examples ofmethods which can be used to analyze the expression level of PBR inconjunction with other genes. For transcriptional profiling, mRNA fromorgan tissue which has undergone ischemia and reperfusion and mRNA fromthe same type of tissue not exposed to ischemia could be reversetranscribed and hybridized to a chip containing DNA from numerous genes,to thereby compare the expression of genes in tissue following ischemiaand normal tissue. By this methodology, complementary indicators may beidentified. For additional methods of transcriptional profiling and theuse of microarrays, refer to, for example, U.S. Pat. No. 6,124,120.

Samples or organ tissue may be obtained during the course of atransplant procedure and post-transplant by any appropriate procedureknown in the art, for example by a biopsy procedure such as by a needlebiopsy.

Where PBR expression levels are observed in at least about 50% or moreof tissue samples, or at least about 70% or more of tissue samples, orat least about 90% or more of tissue samples, this is taken as anindicator of organ health and may be taken as predictive of organviability and/or a favorable outcome. Furthermore, an increased PBRexpression level, preferably a substantially increased level, which maybe observed by immunohistochemistry as intense PBR staining, may also becorrelated with a regeneration process. Thus, where delayed graftfunction is observed, elevated PBR expression may indicate regenerationin the period immediately following transplantation. At later points intime, such as one week or more following transplant, PBR expressionlevels may provide an index of the regeneration progress. For example,an initial increase followed by a subsequent trend towards more normalexpression level has been seen to track the regeneration progress. WherePBR expression levels are substantially reduced or PBR is not detectedin tissue samples, this may be taken as an indicator of mitochondrialdamage and/or of organ damage which may be severe, low organ viability,or as a predictor of a non-favorable outcome.

The method of the invention can be used to determine a discriminatingmarker in the case of non-heart beating donors, for example as a markerof organ viaility. Furthermore, the measurement of PBR expression levelcan be used in conjunction with other indicators of organ health andfunction. Preferably, the methods of the invention will be practiced asa part of the morphological study undertaken after transplantation, suchas kidney transplantation. The index of organ health and recoveryprovided by the methods of the invention may be considered inassociation with other indices and immunomarkers which are used in theclinic.

The following examples of the methods of the invention as applied in ananimal model system are intended to illustrate the invention and shouldnot be construed as limiting the invention in any way.

EXAMPLES

Materials and Methods:

Surgical Procedures and Preservation Solution and Experimental Design:

An art accepted model of cold ischemia and reperfusion injury inautotransplanted pig kidney was used (Goujon et al., Kidney Int.,38:838-50, 2000; Hauet et al., J. Pharmacol. Exp. Ther., 292:54-260,2000; Hauet et al., J. Am. Soc. Nephrol., 11:138-48, 2000). Briefly,following nephrectomy, kidneys were immediately cold-flushed andpreserved at 4° C. for 24, 48 or 72 hours, after which the organs wereautotransplanted. Ureteneocystostomy and contralateral nephrectomy wereperformed. All surgical procedures were performed aseptically. Thepreservation solution was the University of Wisconsin (UW) solution(Ploeg et al., Transplantation, 46:191-96, 1988). The animals weredivided into six groups: control and uninephrectomized (Nef) age-matchedgroup, (n=6, respectively), group UW1 h (UW, 1 h preservation, n=10),group UW24 h (UW, 24 h preservation, n=10), UW48 h (UW, 48 hpreservation, n=10), and group UW72 h (UW, 72 h preservation, n=10).

Renal Function:

Endogenous creatinine (Cr) clearance (C_(cr): ml/min), urine proteinsexcretion and fractional excretion of sodium (FE_(Na): %) were measuredbefore kidney preservation and on postoperative days 1, 3, 5, 7 and 14(D1-D14) and 4 to 12 weeks after autotransplantation (W4-W12). C_(cr),and FE_(Na) were calculated as previously described (10). Na⁺ level wasmeasured by flame photometry and creatinine was measured enzymaticallywith an automatic analyzer (Kodak Ektachem 700 XR, Ortho, Paris,France). Twenty-four-hour protein excretion was measured afterprecipitation by using a colorimetric reaction with pyrogallol(Laboratoire Biorea, Talant, France).

NMR Experiments:

Damage to the renal medulla causes the release in urine of tri-methylamine-N-oxide (TMAO), which is an osmolyte molecule synthesized in therenal medullary cells. Urine and plasma samples from control andpreserved kidneys were studied as described by Hauet et al., (J.Pharmacol. Exp. Ther., 292:254-60, 2000; J. Am. Soc. Nephrol.,11:138-48, 2000). For urine NMR spectra, the ratios of TMAO to Cr werecalculated and expressed in mol/mmol of Cr. To assess proximal tubularinjury, the ratios alanine to Ct concentration were also determined inurine. Citrate is a citric acid cycle intermediate and its reducedexcretion is associated with the impairment of oxidative metabolism andchronic metabolism acidosis. The ratio of the citrate to Cr was alsomeasured in urine and expressed in μmol/mmol Cr.

Histology:

After 1, 24, 48 or 72 h cold storage and 40 minutes of reperfusion, atday 7, day 14 and 4 to 5, and 10 to 12 weeks after surgery, biopsytissue samples from the deep cortex-outer medulla region were performed.Samples were fixed with Dubosq-Brasil and 10% formalin in 0.01mmol/liter phosphate buffer (pH 7.42) and embedded in paraffin.Conventional stains were applied (hematoxylin and eosin, periodicacid-Schiff). Histological analysis concerned proximal tubular cells andparticularly the brush border. Two observers who were unaware of theorigin of the slides reviewed light microscopic studies. Lightmicroscopic sections were examined for tubular necrosis, tubulardilatation, and intratubular detachment. Histological lesions wereexpressed in percent of kidney samples using a previously describedsemi-quantitative scale: 0—no abnormality; 1—mild lesions affecting lessthan 25% of kidney samples; 2—lesions affecting 25-50% of kidneysamples; 3—lesions affecting 50-75% of kidney samples; 4—lesionsaffecting more than 75% of kidney samples.

Immunohistochemistry:

Tubulointerstitial injury was defined as inflammatory cell infiltrates,tubular atrophy or interstitial fibrosis. To estimate the level oftubulointerstitial fibrosis, tissue sections were also labeled withPicro Sirius for collagen identification (collagen I and III). Theamount of interstitial fibrosis was determined in Picro Sirius stainedsections by a semi-quantitative imaging technique. The percentage ofPicro Sirius stained surface was determined on ten different tissuesections viewed at (100 magnification in each experimental condition andexpressed as percent of the total surface area examined). Tubularatrophy, interstitial fibrosis and glomerulosclerosis weresemi-quantitatively scored on a scale of 0 to 4+ by two pathologistsblinded to the experimental conditions (0, normal; 0.5, small changesaffecting 5 to 10%; 1, changes affecting 10 to 25% of specimen area; 2,changes affecting 25 to 50% of specimen area; 3, changes affecting 50 to75% of specimen area; 4, changes affecting 75 to 100% of specimen area).Indirect immunofluorescence using a monoclonal antibody against theswine vimentin (clone V9, MCA862, Serotec Product Data Sheet, Oxford,United Kingdom) was also performed. Immunohistochemistry was performedon paraffin embedded sections. Sections were preincubated with normalgoat serum for 30 minutes and incubated with primary antibody (1:40) for30 minutes at room temperature. Sections were then incubated with thesecondary antibody (rabbit anti-mouse IgG HRP conjugate).Immunolocalization of PBR was determined using an affinity purifiedanti-PBR peptide antiserum raised against an amino acids sequence(amino-acids 9-27, VGLTLVPPSLGGFMGAYFVR) conserved across species(Hardwick et al., Int. J. Cancer, 94:322-27, 2001; Li et al., Proc.Natl. Acad. Sci. USA, 98:1267-1272, 2001; Lacapere et al., Biochem.Biophys. Res. Commun., 284:536-41, 2001; for additional examples ofmethods see, Oke et al., Mol. Cell Endocrinology, 87:R1-R6, 1992;Suarez-Quian et al., Endocrinology, 132:444-58, 1993; Amri et al.,Endocrinology, 137:5707-18, 1996; Hardwick et al., Canc. Res.,59:831-42, 1999). Paraffin embedded sections were incubated with rabbitanti PBR (1:400, dilution with 10% FBS-PBS) for 1 h at room temperature.After rinsing the sections in PBS, horseradish peroxidase conjugatedgoat anti-rabbit IgG (Transduction Laboratory, Lexington, Ky.), diluted1:500. The intensity of immunostaining of vimentin was quantified on a 0to 4+ scale (0=absent staining to 4+=dense). PBR staining was determinedas follows: 100 tubule sections were examined for positive staining andthe intensity of immunostaining was also quantified on a 0 to 4+ scale(0=absent staining to 4+=dense). To test the validity of this scoringsystem, two different observers both of who were unaware of the originof the sections scored all vimentin and PBR stains independently.

Indirect immunocytochemistry was also performed using the human antiCD20 B cell marker which cross reacts with pig B cells (Dako,Copenhagen, Denmark), the mouse anti-pig CD4 (MCA1749; Serotec ProductData Sheet), the mouse anti-pig CD8 (MCA1223), and the mouse anti-pigMC1218 macrophage/monocyte and neutrophils markers (Serotec Product DataSheet) for 30 minutes at room temperature. In all cases, the sectionswere rinsed in PBS and incubated with biotylated antispecies (Dako Ltd,Copenhagen, Denmark) for 20 minutes (1:100) at room temperature. Ascontrols, omitting the primary antibodies, indirect immunofluorescencewas performed. Phosphatase alkaline activity was revealed using freshlyprepared Fas red substrate solution (Sigma, St Louis, Mo., USA) inTris-buffered saline (TBS). Sections were counterstained in hematoxylinand mounted in Aquamount (Gurr, London, UK). All sections were alsoexamined under blind conditions and photographed. The number of CD4,CD8, and MC1218-labeled cells per surface area (10⁴/μm²) was counted onfive different tissue sections in each of the experimental conditions.

Statistical Analysis:

Mean values were calculated for each group (mean±SEM) and compared forstatistical significance by the unpaired t test or variance analysis,and Student-Newman Keuls for multiple comparison tests. The unpaired ttest was used for cellular infiltration and the Mann-Whitney U test wasused for histologic data analyses and immunohistochemical data.Differences at a P value of less than 0.05 were considered to besignificant.

Results:

Effect of Cold Ischemia Time on Renal Function and Survival:

Total body weights and kidneys weight were not significantly differentbetween control (43.2±2.5 kg), Nef (41.3±2.2 kg) and experimental groups(Table 1). Three pigs died on post-operative day 7 and 10 in group UW48h and 6 pigs died on postoperative day 5 and 8 in UW72 h group. Allthese animals developed acute renal failure, confirmed by histologicalanalysis. Survival was 100% in the control group, 100% in the Nef group,UW1 h group and UW24 h. Functional data were not determined in groupsUW48 h and UW72 h (<100 mL/24 h from day 1 to day 5) related to aprolonged anuria before D3 and D7. As shown in FIG. 1, the cold ischemiaand reperfusion affect the renal functions after autotransplantation.C_(cr) was dramatically decreased in UW groups particularly after 48 hcold storage (FIG. 1A) and conversely FE_(Na) was significantly higherin UW preserved kidneys (particularly in groups UW48 h and UW72 h) thanin control group (FIG. 1B). The highest C_(cr) occurred in experimentalUW1 h after autotransplantation, between D1 and W2. There was atransient proteinuria in all-experimental groups, which decreasedprogressively between D1 and W4. Progressive proteinuria developed againafter week 4 following surgery in urine from Nef group and kidneyspreserved and transplanted particularly in groups UW48 h and UW72 h.Proteinuria was significantly lower in UW1 h groups than those coldflushed and preserved for 24, 48 and 72 h in UW solution (FIG. 1C).Aminoaciduria was significantly reduced in Nef and UW1 h when comparedto kidneys preserved 24, 48 and 72 h (FIG. 1E).

Effect of Cold Ischemia Time on Renal Medulla Injury and CitrateExcretion:

TMAO excretion was significantly reduced in UW1 h when compared tokidneys preserved for 24, 48 or 72 h (FIG. 2A). Decreased citrateexcretion was detected in kidneys preserved 24, 48 and 72 h whencompared to other groups between D1 and D7 (FIG. 2B). Citrate excretionremained at a low level in UW48 h, and particularly UW72 h until W4 toW6 where the excretion improved. After W12, excretion of citrate wasmore important in control and strongly reduced in group UW72 h.

Effect of Cold Ischemia on the Morphology of Preserved and ReperfusedKidneys:

After 40-min reperfusion, kidneys flushed and preserved for 72 h with UWsolution showed significantly higher graded score than kidneys flushedand preserved with UW solution for 1, 24 and 48 h (Table 2). Thesedifferences were more pronounced with respect to tubular dilatation,intratubular cell detachment, cast formation and tubular cell brushborder integrity (Table 2). Interstitial fibrosis stained with PicroSirius were significantly reduced two weeks after transplantation inkidneys preserved in UW1 h compared with kidneys preserved 24, 48 or 72h (Table 3). At weeks 4 and 12 following surgery, more interstitialfibrosis was observed in UW24 h, UW48 h and UW72 h groups. The meanaverage score for tubular atrophy, interstitial fibrosis andglomerulosclerosis was also significantly greater in kidneys coldflushed and preserved with UW for 24, 48 and 72 h than those preservedfor 1 hour.

Effect of Cold Ischemia on the Vimentine and PBR Staining:

Vimentin expression was modified by preservation conditions. Theexpression of vimentin was detected 4 weeks after surgery and wasreduced in UW1 h and UW24 h when compared to other experimental groups.The positive reactions were localized mainly in epithelial cells liningdilated or atrophic proximal tubules (FIG. 3). In the present study, thenumber of vimentin-positive renal tubules increased with advancing gradeof fibrosis and reduction of renal function. The immunostaining of PBRin the pig kidney is close to the immunostaining in the human kidney(FIG. 4, Table 4). The number of tubule, which expressed PBR wassignificantly higher in control, Nef and UW1 h groups particularly atD7, D14 and W4. The significantly strongest intensity of PBR wasdetected in UW1 h and UW24 h groups at D7 and D14 (FIG. 5). However theintensity was reduced after D14 in these groups when compared to UW48 hand UW72 h. As expected, PBR was also strongly expressed in regeneratingtubular cells and its expression was modulated by the duration ofpreservation (FIGS. 6A, B, D, E and F). Atrophic tubules surrounded byinterstitial fibrosis did not expressed PBR (FIGS. 6C and G). Thesefindings were consistent with functional data.

Effect of Cold Ischemia on the CD4, CD8, Monocytes and MacrophagesInfiltration:

We have demonstrated in previous studies that T-lymphocytes infiltrationbecome prominent during the early phase following autotransplantation(4,21). This study demonstrated that the preservation in UW solution for1 h reduced the cellular infiltration in autotransplanted pig kidneyswhen compared to other preserved groups (FIG. 7A). As previousdescribed, the number of CD4-positive cells gradually increased from D5to D14, decreased from weeks 2 to 5 and gradually increased from weeks 4to 5 to weeks 10 to 12 in the groups UW24 h and UW48 h. After 72 h coldstorage, the number of CD4⁺-cells increased from D5 to weeks 4 to 5 andfrom weeks 4 to 12 following surgery (FIG. 7A). A biphasic period CD4⁺infiltration occurred after 48 h cold storage and not after 72 h coldstorage. In contrast to that observed with CD4⁺-cells, the number ofCD8⁺-cells increased from D5 to weeks 2 and decreased from weeks 2 to 12following reperfusion in 72 h cold stored kidneys (FIG. 7B). After 48 hcold storage, the number of CD8⁺-cells increased from week 2 to weeks 4to 5 and slightly decreased from weeks 4 to 5 to weeks 10 to 12following autotransplantation. Moreover, the number of CD8⁺-cells wasalso significantly reduced in UW1 h and UW24 h when compared to UW48 hand UW72 h groups. Positive staining using the anti-CD20 B cell antibodyon kidney biopsies taken after reperfusion was never observed (data notshown). Positive staining with the MC1218 macrophage/monocyte wasdetected in all kidney biopsies taken 5 days after transplantation (FIG.7C). However, there were more MC1218-positive cells in posttransplantedkidneys from groups UW48 h and UW72 h. This infiltration ofmacrophage/monocyte disappeared on biopsy samples performed two weeksafter transplantation from 48 h cold-stored kidneys and decreased in 72h cold-stored kidneys. In contrast, MC1218-positive cells were detectedon biopsy samples performed 12 weeks following transplantation. Thenumber of MCA1218-positive cells was much lower in kidneys cold flushedand preserved for 1 and 24 h in UW solution than those preserved with UWfor 48 and particularly 72 h.

TABLE 1 Pigs and kidneys weight in experimental groups: UVV1 h UW24 hUW48 h UW72 h Pig weight (Kg) 46.1 ± 3.1 48.3 ± 2.2 45.7 ± 4.2 48.1 ±3.2 Kidney weight (g) 137 ± 7  139 ± 10  135 ± 9.7 136 ± 13

TABLE 2 Quanitation of morphological data from 48 h cold stored andnormothermic perfused kidneys (Perfusion) and at day 7 and 14:Reperfusion UW1 h UW24 h UW48 h UW72 h Microvilli 0.5 ± 0.1 1.8 ± 0.1*3.5 ± 0.1** 4.0 ± 0.1^(#)** Desintegration Intratubular Cell 0.5 ± 0.12.6 ± 0.1* 3.5 ± 0.2** 3.7 ± 0.2^(#)** Detachment Tubular 0.3 ± 0.2 2.5± 0.1* 3.5 ± 0.2** 3.8 ± 0.2^(#)** Dilatation Cast Formation 0.5 ± 0.12.5 ± 0.1* 3.3 ± 0.2** 3.6 ± 0.2g^(#)** Day 7 Microvilli 0.3 ± 0.1 1.2 ±0.1* 2.7 ± 0.1** 3.3 ± 0.1^(#)** Desintegration Intratubular Cell 0.3 ±0.1 1.1 ± 0.1* 3.2 ± 0.2** 3.5 ± 0.2^(#)** Detachment Tubular 0.3 ± 0.10.7 ± 0.1* 3.0 ± 0.2** 3.5 ± 0.2^(#)** Dilatation Cast Formation 0 0.4 ±0.1* 2.5 ± 0.2** 3.0 ± 0.2^(#)** Day 14 Microvilli 0 0.6 ± 0.1 2.5 ±0.2** 2.9 ± 0.2 Desintegration Intratubular Cell 0 0.5 ± 0.1 2.9 ± 0.3**3.2 ± 0.3 Detachment Tubular 0 0.6 ± 0.1 2.5 ± 0.2** 3.1 ± 0.2Dilatation Cast Formation 0 0 0.7 ± 0.2** 1.2 ± 0.2 *P < 0.05 UW72 h, 48h, 24 h vs UW1 h **P < 0.01 UW72 h, 48 h, 24 h vs UW1 h ^(#)P < 0.05UW72 h vs UW48 h ^(##)P < 0.01 UW72 h vs UW48 h

TABLE 3 Quantitation of tubular atrophy, interstitial fibrosis andglomerulosclerosis: Groups Week UW1 h UW24 h UW48 h UW72 h Tubularatrophy (%) 2 1.8 ± 0.4  5.4 ± 1* 18.7 ± 1.7*** 26.5 ± 3^(#)*** 4-5 2.5± 0.5  8.5 ± 1.5** 21.5 ± 2.4*** 34.8 ± 3.4^(#)*** 10-12 2.9 ± 0.6 10.2± 2** 28.6 ± 3.2*** 42.5 ± 4.1^(#)*** 16  3.2 ± 0.6 11.5 ± 2.4** 35.4 ±3.5*** 59.5 ± 5.2^(#)*** Interstitial fibrosis (%) 2 4.2 ± 1.0  8.2 +1.1* 12.6 ± 1.7***   26 ± 3.0^(#)*** 4-5 5.1 ± 1.8 11.4 ± 1.5*   17 ±2.4***   33 ± 3.8^(#)*** 10-12 7.5 ± 2.2 16.2 ± 1.5*   26 ± 3.2***   45± 4.6^(#)*** 16  9.1 ± 3 21.5 ± 2.4**   32 ± 3.5***   65 + 5.0^(#)***Glomerulosclerosis (%) 2 0  8.5 ± 3.2 19.8 ± 1.9 29.8 ± 3.4^(#) 4-5 1.5± 0.4 12.5 ± 2.3** 23.5 ± 2.9*** 34.7 ± 4.0^(#)*** 10-12 1.9 ± 0.3 16.3± 2.5** 32.6 ± 3.2***   46 ± 5.0^(#)*** 16 2.2 ± 0.4 17.9 ± 2.7** 39.4 ±3.5*** 62.8 ± 4.9^(#)*** *P < 0.05 UW72 h, 48 h, 24 h vs UW1 h **P <0.01 UW72 h, 48 h, 24 h vs UW1 h ***P < 0.01 UW72 h, 48 h, 24 h vs UW1 h^(#)P < 0.05 UW72 h vs UW48 h

TABLE 4 Quantitation of PBR expression on tubule and intensity of PBRstaining: Groups Week UW1 h UW24 h UW48 h UW72 h PBR 2 85 ± 2.4 76 ± 3.436 ± 3.4 15 ± 21^(#)** expression 4-5 88 ± 3.3 79 ± 2 37 ± 4.5 17 ±2^(#)** on tubule (%) 10-12 90 ± 4 82 ± 2.2 40 ± 4.1 19 ± 3^(#)** 16  94± 4.1 85 ± 3.2 45 ± 4.1 21 ± 3^(#)** Staining 2 +++/++++ +++ +/++ 0/+intensity 4-5 ++ +++ ++ + of PBR (+) 10-12 ++ +++ ++/+++ +/++ 16  ++/+++++ +++ +++/++++ *P < 0.05 UW72 h, 48 h, 24 h vs UW1 h **P < 0.01 UW72 h,48 h, 24 h vs UW1 h ^(#)P < 0.05 UW72 h vs UW48 h ^(##)P < 0.01 UW72 hvs UW48 h

REFERENCES

The following publications, as well as all others referenced in thedisclosure, are incorporated herein by reference in their entirety:

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For routine practice of the protocols referenced herein, one of skill inthe art is directed to the references cited in this application as wellas the several Current Protocol guides, which are continuously updated,widely available and published by John Wiley and Sons, (New York). Inthe life sciences, Current Protocols publishes comprehensive manuals inMolecular Biology, Immunology, Human Genetics, Protein Science,Cytometry, Neuroscience, Pharmacology, Cell Biology, Toxicology, andNucleic Acid Chemistry. Additional sources are known to one of skill inthe art.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention.

1-15. (canceled)
 16. A method for treating a patient during kidneytransplantation therapy, the method comprising administering aneffective amount of a pharmaceutical composition comprising an agentwhich modulates the activity of peripheral-type benzodiazepine receptor(PBR).
 17. The method of claim 16, wherein progress of the treatment ismonitored by measuring an index of organ health for a transplant kidney,further comprising measuring an expression level of peripheral-typebenzodiazepine receptor (PBR) in the transplanted organ whereby saidindex is determined, wherein the expression level of PBR in the kidneyis correlated with an assessment of kidney health.
 18. An improvedmethod for investigating transplant therapy techniques in animals, theimprovement comprising measuring an expression level of peripheral-typebenzodiazepine receptor (PBR) in a transplanted or autotransplantedkidney and correlating the expression level of PBR in the kidney with anassessment of kidney health. 19-26. (canceled)
 27. A method for treatinga patient during kidney transplantation therapy, the method comprisingadministering an effective amount of a pharmaceutical compositioncomprising an agent which modulates expression of peripheral-typebenzodiazepine receptor (PBR) in a transplanted kidney.
 28. A method oftreating a patient during kidney transplantation therapy, the methodcomprising administering an effective amount of a pharmaceuticalcomposition comprising an agent which modulates the activity ofperipheral-type benzodiazepine receptor (PBR) in a transplanted kidney.29. The method of claim 28, wherein progress of the treatment ismonitored by measuring an index of kidney health comprising measuring anexpression level of PBR in the kidney and correlating the expressionlevel of PBR in the kidney with an index of kidney health. 30.(canceled)